Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

Here, terminating ribosome with a stop codon in the A-site (denoted as R here) binds release factor (denoted as F) with rate constant k1 forming release factor prebound complex R·F from which F can either dissociate with rate constant q1 or recognize stop codon with rate constant kr forming complex R·Fr. Complex R·Fr can either return back to R·F with date constant qr or proceed forward with rate kc to complex R·Fc in which release factor F has undergone a conformation change that leads to the ester bond hydrolysis in peptidyl-tRNA with rate constant kH and peptide release. THB (T) can bind both to R forming complex R·T and to R·F forming complex R·T·F. These THB binding reactions are described by rate constants k2, q2, k4, and q4 as indicated. (b) Termination in Thermorubin (THB) absence (the same as (a) except that THB-containing complexes are omitted). (c) Termination for the case of persistent THB presence. The same as (b) except that THB is assumed to be bound to all complexes on the termination pathway. (d) Termination in the presence of THB. The same as (a) except that simultaneous THB and RF1/2 pre-binding to termination complex R is assumed impossible (i.e., it is assumed that R·T·F complex cannot be reached).
Supplementary Fig. 15. Effect of THB on the splitting of post-termination ribosome complexes (post-TC). (a) Post-TC (0.25 µΜ) was pre-incubated with indicated THB concentrations after which the time course of their splitting into subunits by RRF (20 µΜ), EF-G (10 µΜ), and IF3 (1 µΜ) was monitored by Rayleigh light scattering at 365 nm in stoppedflow. (b) Dependence of the fraction of non-split ribosomes on THB concentration in (a); solid line here is the linear fit of the data with error bars indicating SEM of the data. (c) Post-TC (0.25 µΜ) was pre-incubated with 10 µΜ THB after which the time course of their splitting into subunits by RRF (added in indicated concentrations), EF-G (10 µΜ), and IF3 (1 µΜ) was monitored in stopped-flow. Solid lines in (a) and (c) represent the double exponential fits of the data. Experiments were conducted in triplicates. Supplementary Fig. 16. Pipeline for Cryo-EM data processing. Cryo-EM data was processed using CryoSPARC and the model building was performed using Coot. Fourier Shell Correlation (FSC) analysis is presented in the bottom right (blue -unmasked and orange-masked half maps). The final average resolution of the highly homogeneous THBbound 70S IC, determined on the basis of the FSC value of 0.143 (orange curve) is 1.96 Å. Supplementary Fig. 17. Close look into the THB binding site at the subunit interface. (a) Cryo-EM map fitted to model and (b) map without model depicting bound THB (blue) at the interface between H69 (orange) and h44 (green) of 23S and 16S rRNA respectively. Bound fMet-tRNA fMet (saddle brown) and mRNA (yellow) are also seen clearly. The same contour level was used throughout the figures including tRNA, mRNA, rRNA, and THB, which shows the matching density of all elements in the THB binding pocket of the ribosome. Supplementary Fig. 18. Comparison of our cryo-EM structure of E. coli ribosome-bound THB in the presence of P-site tRNA (8AYE, blue) with the earlier crystal structure of THB-70S (T. thermophilus) complex (4V8A, grey). The superposition of the two structures in four different orientations shows slightly different conformation of the orthohydroxyphenyl group of THB. The difference can be due to THB binding to functional (current) vs. nonfunctional (earlier) ribosomal complex. The difference can also be due to difference of the ribosomes from the two bacterial species. Supplementary Fig. 19. Illustration depicting map-quality of cryo-EM reconstruction of THB bound 70S ribosome (this study) (PDB 8AYE). (a-b) Examples of modifications of rRNA bases in both 23S and 16S rRNA are shown as sticks with overlaid EM densities shown as mesh. In (b) close-up density for Mg ++ with coordination of water/oxygen molecules can be seen. (c) Example EM-densities showing side chain conformations of amino acids in ribosomal proteins including L3, L20, and S5. (d) EM-density of THB binding site with surrounding residues and Mg ++ . The numbers in the brackets indicate the map threshold.

Supplementary Fig. 20. Superimposition of THB-bound 70S ribosome (this study) with the EF-G bound pre-translocation ribosome (PDB 7SSL).
Overlay of these two structures shows no steric hindrance for EF-G binding in the presence of THB in the ribosomal A site. Supplementary Fig. 21. Close-up view of the A-site of 70S ribosome bound to thermorubin (blue) (PDB 8AYE), aminoglycoside paromomycin (brown) (PDB 7K00), and tuberactinomycin viomycin (yellow) (PDB 6LKQ). Important 16S and 23S rRNA nucleobases interacting with these antibiotics are displayed. Cβ outliers (%) 0.00

Supplementary Note 1: Initiation and Complex formation kinetics
The initiation stage of translation cycles starts with the formation of 30S pre-initiation complex (30S pre-IC), which involves binding of IF1, IF2, and IF3 plus mRNA followed by fMet-tRNA fMet binding. Thus, 30S pre-IC contains all three initiation factors and a P-site bound fMet-tRNA fMet that interacts with AUG codon of mRNA in the P-site of the 30S subunit 1,2 . Formation of a 70S ribosome or 70S initiation complex (70S IC) occurs by the association of 50S subunit with naked 30S subunit or complete 30S pre-IC (30S-PIC), respectively 1,3 (as illustrated in Supplementary Fig. 1a). The complete 30S pre-IC is used in the experiments in Figure 2a, while 30S pre-IC formed without IF3 is used in the experiments shown in Supplementary Fig. 2. fMet-tRNA fMet can also bind directly to mRNA programed 70S ribosomes (as in experiments in Figs. 2c and d) through a "non-orthodox" initiation pathway 4 . Such a binding is described by the kinetic scheme shown in Supplementary Fig. 1b. The kinetic schemes shown in Supplementary Figs. 1a and 1b can be viewed as particular cases of a generic "complex formation" scheme in Supplementary Fig. 1c. The kinetic equations that govern the dynamics of this generic "complex formation" scheme ( Supplementary Fig. 1c) are discussed below.

Kinetics of complex formation and dissociation.
Denoting A·B as C the differential equation describing the dynamics of C-concentration in Supplementary Fig.1c is: Here a0 and b0 are total (free plus in C-complex) while a and b are current concentrations of A and B; c is the current concentration of complex C and K=q/k is the equilibrium dissociation constant of complex formation. The above equation can be re-written as: Here, r0 and p0 are the two roots of quadratic polynomial of c: Integrating Supplementary Eq. (2) one obtains (assuming that r0 > p0) : Here we denoted: 0 0 In the case when one studies complex formation by mixing A and B at time zero, c(0)=0 so that 0 0 / p r Ω = . Using this, Supplementary Eq. (4) simplifies to: In the case when a0>>b0 and K>>b0 the roots r0 and p0 can be readily estimated and Supplementary Eq. (6) reduces further to a well -known equation for complex formation kinetics: It shows that by fitting c(t) to an exponential function at different a0 values (initial Aconcentrations) one can recover both the association and dissociation rate constants k and q, respectively 5 . When K is very small in comparison with both a0 and b0, as in all association experiments in Fig. 2 and Supplementary Fig. 2, the r0 and p0 roots approach a0 and b0, respectively. In this case: If, in addition 0 0 a b ≈ , the above relation further simplifies to 3 :

Kinetic of complex dissociation in THB "chase "experiment
The kinetics of THB chase experiment can be described by a kinetic scheme shown in Supplementary Fig. 8. Here, Thermorubin (THB), denoted as T in the figure, is first preincubated with a small excess of ribosomes (denoted as R here) to assure the formation of R·T complex. It can also be assumed (from measured equilibrium dissociation constant of R·T formation) that nearly all THB is bound in this R·T complex. The chase experiment in Supplementary Fig. 9 starts with the addition of a high concentration of another antibiotic, Arbekacin (denoted as A in Supplementary Fig. 8) that competes with THB for the same binding site on the ribosome 6 . One then monitors the dissociation of THB from ribosome as a nearly exponential reduction of fluorescence (Supplementary Fig. 9) because the re-binding of dissociated THB back to the ribosome is blocked by fast Arbekacin binding to the THBfree ribosomes. The formation and dissociation of R·T and R·A complexes is governed by two differential equations: Here, cRA and cRT are the current concentrations of R·A and R·T complexes, respectively; k1 and q1 are the association and dissociation rate constants for Arbekacin binding, respectively; k2 and q2 are those for Thermorubin binding. The time evolution of drug-unbound ribosome concentration cR and free THB can be deduced from conservation laws for ribosomes and THB, respectively: To obtain the kinetic of R·T concentration we use that k1 and q1 for Arbekacin are larger than k2 and q2 for THB and, in addition, that the Arbekacin concentration is much larger than that of THB. This implies that R·A complex equilibrates with free ribosomes very fast (on the time scale of THB dissociation) so that the following relation between the current R and R·A concentrations is valid: From this and the 'matter conservation law' applied to the ribosomes, it follows that: Using the last expressions in Supplementary Eq. (13) and (11), one can re-write Supplementary Eq. (10) for complex R·T concentration as: Here, we introduced for compactness: [ ] Following the same approach as in the previous section one obtains the following solution of Supplementary Eq. (14): Here, r0 and p0 are the two roots of quadratic polynomial in cRT: Like before (see Supplementary Eq. (6)) 0 0 In the chase experiment we use Arbekacin concentration sufficiently high to ensure that w in Here: It shows that fitting experimental data on fluorescence decreases in Supplementary Fig. 9 to Supplementary Eq. (19) one obtains rate Q that is always larger than genuine THB dissociation rate q2. In other word, Q is the upper bound for q2. Moreover, Supplementary Eq. (20) shows that the higher is the chaser A concentration the closer Q is to q2.

Supplementary Note 2: Elongation phase of translation and its "meantime" analysis
Elongation cycle of the ribosome is depicted in Supplementary Fig. 4a. The cycle starts with a post-translocation ribosome or with its mimic, a 70S Initiation complex (IC) containing an mRNA codon in otherwise vacant A-site (denoted here as RA). To this ribosome RA a ternary complex Tu·GDP dissociates fast with the rate constant kTu·GDP leading to a complex denoted here as R•Nt with non-accommodated aa-tRNA sitting in the A-site of the 30S subunit. Then aa-tRNA accommodates with rate kA into the A-site on the 50S subunit leading to a complex denoted here as R•t. During the accommodation aa-tRNA can dissociate with the rate kaa-tRNA from the ribosome in a so-called proofreading reaction also re-generating RA ( Supplementary  Fig. 4a). When post-accommodation complex R•t is reached, the accommodated A-site aa-tRNA accepts the peptide from the P-site tRNA in a peptidyl transfer (PT) reaction that occurs with rate kp leading to complex R•P in which a newly formed A-site peptidyl-tRNA carries a peptide extended by one amino acid. To this R•P complex EF-G (G) binds forming complex R•P•G. The bound EF-G makes the new peptidyl-tRNA to translocate from the A-to P-site of the ribosome with rate kT leading back to the ribosome RA with extended peptide and a new A-site codon in otherwise empty A-site ready to bind a new ternary complex.
Importantly, EF-Tu· GDP dissociated from complex 3 D R T ⋅ requires GDP to GTP exchange factor EF-Ts to convert EF-Tu· GDP back to EF-Tu· GTP.
It should be noted that the elongation cycle in Supplementary Fig. 4a is somewhat simplified in that the step of T3 pre-binding to the ribosome and the subsequent step of codon: anticodon recognition that leads to GTP hydrolysis on EF-Tu 7,8 are lumped here together. We have further assumed that after translocation EF-G dissociates from the post-translocated ribosome very fast so that a short-lived complex RA•G can be omitted.

Single cycle kinetic experiments with elongating ribosome.
The ultimate aim of single cycle experiments is to determine the rate constants of kinetic schemes or, failing to determine all rate constants, to extract some of them or their combinations. The kinetic scheme of elongation cycle in Supplementary Fig. 4a can describe both multi-cycle and single cycle experiments depending, for example, on availability of T3 with a cognate aa-tRNA able to read a new codon in the RA ribosome (the one to the right in Supplementary Fig. 4a) or availability of EF-G. A curtailed, "single cycle" version of elongation cycle for the case when EF-G is excluded is depicted in Supplementary  Fig. 4b. In a corresponding single cycle experiment 9 one usually monitors the time course of accumulation of complex R•P that contains the extended peptide (dipeptide in case when RA is a 70S initiation complex). After mixing RA ribosomes and ternary complexes 3 T T at the start of experiment the ribosome RA transits through several complexes as time passes by ending up in complex R•P (see Supplementary Fig. 4b) from which it cannot move further (because of EF-G omission). R•P accumulation can be monitored (with the help of HPLC) as the time course of the formation of peptide with the length increased by one amino acid 9 .
The dynamic of concentrations of RA ribosomes, different ribosomal complexes, free 3 T T , dissociated Tu·GDP and aa-tRNA in Supplementary Fig. 4b is governed by the following set of differential equations: Here, cRA is the current concentration of RA ribosomes; cT3 is that of 3 with non-accommodated aa-tRNA while cRt is that of R·t complex with accommodated aa-tRNA and cRP is the concentration of R·P complex containing the extended peptide. All these concentrations are, of course, the functions of time. In addition to the accumulation of extended peptide one can also monitor (in the same experiment) the accumulation of GDP in 3 D R T ⋅ complex and in Tu·GDP dissociated from this complex 10,11 .
Importantly, a GDP to GTP exchange factor EF-Ts that converts Tu·GDP back to Tu·GTP is excluded in such experiments to assure that GDP remains on EF-Tu after GTP hydrolysis for a sufficiently long time, about 3 min, to keep reacted EF-Tu inactive and unable to bind aa-tRNA for the whole duration of single cycle experiment 10,11 . T . This leads to:

Mean times of GTP hydrolysis and peptide bond formation
Here, we formally introduced notations X Θ for the integrals: We will see later that some of these integrals can be identified as mean life-times of the corresponding complexes. Appearance of several 1 and 0 on the left side in Supplementary Eq. (22) is because upon integration we obtain Here, F is a so called proofreading factor 12 also obtained from Supplementary Eq. (22) solution as: Further, it is easy to see from the law of matter conservation applied to EF-Tu that: Dividing both parts of this relation by cT3(0) and integrating one obtains: Here we used definitions of Here: { } , that the reaction will proceed to the product, GDP, from this complex 9,12 . We note that Supplementary Eq. (29) holds also for more detailed kinetic schemes in which T3 pre-binding and codon recognition steps are considered separately 8

Di-peptide formation
To see how one obtains mean time of extended peptide formation, τEP, called τDip in the main text in experiments in Fig. 3b, we apply the law of matter conservation to aa-tRNA that was initially in T3. It requires that: To obtain the current concentration of aa-tRNA discarded at the proofreading step (during accommodation) we consider three last equations in Supplementary Eq.  25)) one obtains after some algebra: Integrating both parts of this relation one finds that: The integral on the left side in Supplementary Eq. (34) can be interpreted as a mean time τEP of peptide extension by one amino acid in single cycle experiment 9 . In experiments in Fig.  3B we start with 70S IC in place of RA so that peptide extension results in dipeptide formation, and we will use τDip instead of τEP here to conform with the main text notations.  Supplementary Fig. 4b). The first term describes a very fast Tu• GDP dissociation from 3 D R T ⋅ complex and can be neglected, so that subtracting τGTP from τDip one obtain the sum 1/ (kA+ kaa-tRNA)+1/kP that gives the low bound for the sum 1/kA+1/kP of mean times of aa-tRNA accommodation and peptidyl transfer 10,11 .

Why do we fit the time courses of GTP hydrolysis or Dipeptide formation by a weighted sum of exponential functions?
We first note that when ribosome concentration is much higher than that of Here, the number of exponents is equal to the number of complexes in the scheme and λivalues are related (sometimes through rather involved algebra 13 ) to the rate constants of the kinetic scheme in Supplementary Fig. 4b. Normally, many λi-values are much larger than others and their weights are small. That explains why one rarely fits the experimental dipeptide formation curve cRT(t) (denoted usually as cDip(t) ) using more than 2-3 weighted exponentials. In practice, cDip(t) curve is normally fitted using a "two-exponential" fit equation for a two-step reaction 5 : Another advantage of "exponential curve fitting" over direct numeric integration is that the fitting supplies errors of λi-parameters in Supplementary Eq. (36) from which the error in mean time τDip can be extracted 11 . We note that in the case of GTP hydrolysis the single exponential fit: is normally used because the second term with kH in Supplementary Eq. (28) is much smaller than the first term and can be neglected 9 . Importantly, this also implies that in the case of GTP hydrolysis λ-parameter is given by: can be obtained as λ/cRA.

Single cycle experiments for the case of large excess of T3 over RA ribosomes.
We again consider kinetic scheme in Supplementary Fig. 4b, but assume a large excess of ternary complexes T3 over RA ribosome as in experiments in Figure 3C. In this case we can assume T3 concentration constant, exclude the second equation in Supplementary Eq. (21) (since we are not interested in free T3 dynamics) and integrate both sides of the equations describing the dynamics of ribosome complexes from the start of the reaction (time 0) to the time when all RA ribosomes ended up in complex R•P (we denote this time as infinity, ∞). This time, we will, however, normalize the result of integration by the total concentration of ribosomes equal to cRA(0) . One obtains: ( ( ) ( )) 1 1 1 The main differences between the two cases is that in the former case aa-tRNA discarded from R•Nt in the proofreading step cannot be re-used again, while in the latter case the ribosome RA discarded in the same proofreading step has another chance to bind a new T3 to go through the same complexes again and again (F-times on average) until it finally reaches R•t and spends time 1/kP to make a peptide bond. This Ftime cycling of I until its arriving to R•t complex explains why mean life times of complexes before complex R•t (see Supplementary Fig. 4b) are F-fold longer in Supplementary Eq. (44) compared with those in Supplementary Eq. (35).

Formal definition and explanation of mean times meaning
It is easy to see that we can interpret the ratio cX(t)/ cRA(0) in Supplementary Eq. (41) as the probably PX(t) to find the ribosome in complex X at time t, i.e. : Here, total ribosome concentration cTot is the same as cRA(0). Further, the time integral over PX(t) have the meaning of time the ribosome spends (on average) in complex X during single cycle experiment. To see this, we cover the time axis from 0 of experiment start to time T of experiment duration by intervals ∆tj with centers at tj.
is the average time the ribosome spends in complex X during an experiment of duration T. Now, the time it spends in complex X tills the experiment is assuredly over will be: This time τX is often called a "mean (life) time" of complex X (on the reaction pathway).

Extraction of translocation time from two-or three-peptide bond formation experiment.
In the two-or three-peptide bond formation experiments one allows the ribosome to go through 2 or 3 elongation cycles depicted in Supplementary Fig. 4a and accumulate peptide extended by 2 or 3 amino acids (Figs. 3d, 3e, 3f). This is achieved by using mRNA with an appropriately positioned stop codon. In this work we use 70S initiation complex (70S IC) as an RA post-translocation ribosome analogue so that the two amino acid peptide extension experiment analyzed below can be called a tri-peptide formation experiment. The analysis will demonstrate that the mean time of tri-peptide formation is the sum of mean times of two peptide extensions plus mean time of translocation reaction. This assertion is usually based on the analysis of a three step kinetic scheme depicted in Supplementary Fig. 4c 14 .
We will, however, demonstrate the validity of this important assertion for the complete kinetic scheme of elongation cycle depicted in Supplementary Fig. 4a in which ribosome goes through two complete elongation cycles and stops because of RA encountering a stop codon. The analysis below can be readily extended to any number of cycles. The dynamic of concentration of ribosome complexes for the kinetic scheme in Supplementary Fig. 4a that is allowed to go for two cycles under condition of a large T3 and EF-G excess over ribosomes is governed by the following set of differential equations for the first and second cycles of peptide extension: ( (2) (2) (2) We first note that the time course of the accumulation of two-amino acids extended peptide is given by the sum (2) (2) because we do not discriminate between preand post-translocated peptide of the same length in quench flow experiments. For brevity we and call it a current tri-peptide concentration.

t c t c t c t c t c t c t c t c t c t c t c t
The mean time of two-amino acid extended peptide (tri-peptide) formation τTrip is then: The first term in Trans τ to the right correspond to the mean time of EF-G binding to pretranslocation ribosome and the second term is the mean time of translocation by ribosome bound EF-G 14 . This explains why the mean translocation time (at a given EF-G concentration) can be obtained from tri-peptide experiment by subtracting two mean times of di-peptide formation. Similar analysis for the case of tetra-peptide shows that: In is important to point out that mean time of di-peptide formation can be extracted directly from tri-peptide formation experiment excluding the necessity of a separate di-peptide experiment. This is because when monitoring the time course of tri-peptide accumulation by HPLC one also simultaneously monitors the time course of di-peptide 15 . In the case of dipeptide one actually monitors the sum, cDip(t) of di-peptides in all ribosomal complexes that contain di-peptide, i.e. : (1) (1)

t c t c t c t c t c t c t
It is easy to see then that:

c t c t c t c t c t c t
Thus, we can extract τDip from the time evolution of cDip(t) +cTrip(t) 15 .
We note that tripeptide formation curve ( ) Trip c t is usually fitted using "three step kinetic scheme equation" 15 :  4c).

Relation between "single cycle" and steady-state Michaelis-Menten kinetics.
It is often asked how the kcat, KM and kcat/KM parameters pertaining to the realm of steadystate Michaelis-Menten kinetics can be obtained from "single-cycle" pre-steady-state experiments. To see the connection between "single-cycle" and Michaelis-Menten kinetics we first notice that the net result of one elongation cycle in Supplementary Fig. 4a is the peptide extension by one amino-acid which can be also viewed as a one peptide bond addition cycle by a ribosome-enzyme. We can thus consider the kinetic scheme in Supplementary Fig. 4a as the scheme describing transitions of initially free enzyme RA (which is ribosome RA to the left) to its first bound state  Supplementary Fig. 4a.
The mathematical equivalence of single-cycle equations for τX and steady state equations for cX/j is quite general and can be demonstrated for many kinetic schemes that can operate both in single-cycle and multi-cycle modes.

Supplementary Note 3: Termination phase of translation cycle and termination kinetics
The termination phase of translation cycle is depicted in Supplementary Fig. 14a. Here, terminating ribosome (R) with stop codon in the A-site binds release factor (F) with rate constant k1 forming release factor pre-bound complex R·F from which F can either dissociate with rate constant q1 or recognize stop codon with rate constant kr forming complex R·Fr.
Complex R·Fr can either return back to R·F with date constant qr or proceed forward with rate kc to complex R·Fc in which release factor F undergone a conformation change that positions its GGQ loop into the PTC of the ribosome. This leads to the ester bond hydrolysis in peptidyl-tRNA with rate constant kH and peptide release 16 . THB (T) can bind to R forming complex R·T and it can bind to R·F forming complex R·T·F. These THB binding reactions have rate constants depicted in Supplementary Fig. 14a. The kinetics of concentrations of different complexes in Supplementary Fig. 14a The meantime, P τ of peptide release is then the sum of all mean times in Supplementary Eq.
(65), which we compact to: Here kcat/KM and kcat of the release reaction described by Supplementary Fig. 14b (67)).

Mean-time of peptide release in THB presence when THB does not dissociate from terminating ribosome
In the release experiments conducted in THB presence, THB was first pre-incubated with terminating ribosomes before release factor F addition. In this case all terminating ribosomes (R) are initially THB bound in complex R·T. Let us first consider a kinetic scheme of release reaction in which THB, once bound to R, remains bound all the way through the reaction until peptide is released. The kinetic scheme describing this situation is depicted in Supplementary Fig. 14c. This scheme is formally identical to that in Supplementary Fig. 14b except for different names for complexes and rate constants. The "mean-time treatment" of this scheme is, therefore, identical to that of scheme in Supplementary Fig. 14b From structural consideration it is reasonable to assume that THB presence on the terminating ribosome will severely inhibit the codon recognition by release factor, i.e., that rate constant THB r k will be greatly reduced and THB r q greatly increased due to THB presence. We note that termination scheme in Supplementary Fig. 14a Supplementary   Fig. 14c and all other rate constants are as in Supplementary Fig. 14b we can use Supplementary Eq. (69) to obtain the following expressions for the inverses of kcat/KM and kcat parameters: (

Mean-time of peptide release when THB dissociation from ribosome is required for the release
Let us now assume that THB and RF1/2 cannot pre-binding to terminating ribosome R simultaneously so that R·T·F complex cannot be reached and THB dissociation from R·T complex is required for the release factor recognition of stop codon and peptide release. This reduces the scheme in Supplementary Fig. 14a to the scheme in Supplementary Fig. 14d The peptide release scenario described by Supplementary Fig. 14d explains, therefore, a drastic decrease in effective kcat (due to a long time 1/q2 of THB dissociation) and a drastic decrease in effective kcat/KM (due to small K2 for THB binding) in THB presence (see Supplementary Eq. (75)). However, Supplementary Eq. (74) predicts a strong τP increase with increasing THB concentration at a fixed release factor concentration not observed experimentally (see Supplementary Fig. 13b). Thus, the termination scheme in Supplementary Fig. 14d in which R·T ·F complex cannot be formed fails to explain our experimental results in Supplementary  Fig. 13b.
Let us now consider the termination scheme depicted in Supplementary Fig. 14a Supplementary Eq. (85) shows that under assumption that the pre-binding of release factor in R·T·F complex greatly destabilizes THB binding to it, Supplementary Fig. 14a can explain both great decrease in kcat and kcat/KM as well as the absence of their dependence on THB concentration. Hence, kinetic scheme of termination in Supplementary Fig. 14a can also explain all experimental results in Fig. 4. We note also that if the release factor pre-binding in R·T·F complex had no effect on the rate constant of THB dissociation, i.e., if q4 = q2 then Supplementary Eq. (82) transforms into: